Overexpression and knockdown of CDKN2A were performed in human glioma cell lines.. Results: Here we show that a lower expression of CDKN2A and a higher expression of cyclin D1 in the pat
Trang 1R E S E A R C H Open Access
Downregulation of CDKN2A and suppression of cyclin D1 gene expressions in malignant gliomas Weidong Liu, Guohua Lv, Yawei Li, Lei li and Bing Wang*
Abstract
Background: Malignant gliomas are the most common in central nervous system cancer Genome-wide
association study identifies that CDKN2A was a susceptibility loci for glioma The CDKN2A/cyclin-dependent kinase
4, 6/Retinoblastoma protein (Rb) pathway is thought to play a crucial role in malignant gliomas pathogenesis We have investigated the expression of CDKN2A for potential correlations with malignant gliomas grade and potential role of CDKN2A on malignant gliomas pathogenesis
Methods: Tumour tissue samples from 61 patients suffering from malignant gliomas were investigated The
expression levels of CDKN2A were detected using immunohistochemical staining and western blot Overexpression and knockdown of CDKN2A were performed in human glioma cell lines Subsequently, colony formation, growth curves and CDKN2A-Cyclin-Rb pathway were analyzed
Results: Here we show that a lower expression of CDKN2A and a higher expression of cyclin D1 in the patients with high-grade malignant gliomas than low-grade gliomas, respectively Moreover, overexpression of CDKN2A inhibits growth of glioma cell lines by suppression of cyclin D1 gene expression
Conclusions: Our study suggests that CDKN2A as a malignant gliomas suppressor gene, appears to be useful for predicting behaviour of high-grade malignant gliomas CDKN2A-Cyclin-Rb pathway plays a key role on malignant gliomas formation and that therapeutic targeting of this pathway may be useful in malignant gliomas treatment
Background
Glioma is the most frequent primary intracranial
tumour in both adults and children Their incidence
rate is about 6.42 cases/100,000 [1] The molecular
genetic alterations with the development and
pathogen-esis of human gliomas have been widely studied [2]
Germline mutations, somatic mutation, disruption, copy
number variation of genes and loci contribute to the
pathogenesis of glioma [3-7] Genetic alterations
fre-quently involved, include amplification of genes
encod-ing for receptor tyrosine kinases (EGFR, PDGFRA),
onocogens (PDGF, PDGFR, CDK4) and
deletions/muta-tions in tumor suppressor genes (IDH1, IDH2, TP53,
CDKN2A, PTEN)[6,8] In recent years, the molecular
understanding of glioma has greatly increased
Activa-tion of the MAPK/ERK and PI3K/AKT pathways are
hallmarks of a variety of malignancies, including
mela-noma and high-grade astrocytomas [6] CDKN2A, a
tumor suppressor protein, has been shown to block MDM2-induced degradation of p53 and enhancing p53-dependent transactivation and apoptosis CDKN2A also binds to CDK4 and CDK6 and suppresses proliferation
by inhibiting cells progressing from G1 into S phase [9]
We reported that expression of CDKN2A (encoding p16 protien) was lower in the patients with high-grade malignant glioma than low-grade glioma Moreover, overexpression of CDKN2A inhibits growth of glioma cell lines by suppression of cyclin D1 gene expression
Methods
Tissue samples and cell lines
A total of 61 patients with malignant glioma were included in this study All patients underwent surgery at Xiangya Secondary Hospital during the period
2009-2010 in accordance with China law and ethical guide-lines, and informed consent was obtained from patients prior to resection Glioma cells (T98G, U251-MG,
U87-MG, A172, SW1736, U118-U87-MG, U138-U87-MG, H4 and HS-683) were purchased from ATCC and were cultured in
* Correspondence: wang_bing2011@yahoo.com.cn
Department of Spinal Surgery, Second Xiangya Hospital, Central South
University, 139 RenMin Road, Changsha, China
© 2011 Liu et al; licensee BioMed Central Ltd This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
Trang 2Dulbecco’s modified Eagle’s medium (GIBCO)
supple-mented with 10% fetal bovine serum (GIBCO) and 4
mM glutamine
Immunohistochemistry
Paraffin-embedded sections were deparaffinized and
subjected to immunohistochemical staining for
CDKN2A with CDKN2A monoclonal antibody (Cell
Sig-nal Technology) The sections were microwaved in 10
mM sodium citrate buffer (pH 6.0) at 10 min intervals
for a total of 20 min Endogenous peroxidase activity
was blocked by incubating the sections in a solution of
3.0% hydrogen peroxide for 20 min at room
tempera-ture After washing in PBS the sections were incubated
with the primary CDKN2A monoclonal antibody
(1:100), overnight at 4°C The sections were washed
with PBS and incubated with biotinylated secondary
antibody for 30 minutes, followed by incubation with
streptavidin-biotin-peroxidase complex a solution
3-3’diaminobenzidine (Sigma), containing 1.0% hydrogen
peroxide and lightly counterstained with Harris
hematoxylin
Western blot
Tissues form patients were homogenized with lysis
buffer containing 50 mM Tris-HCl, 150 mM NaCl, 1%
sodium deoxycholate, 0.1% SDS, 20 mM EDTA, 1 mM
NaF, and 1% Triton X-100 (pH 7.4) with protease
inhi-bitors (Sigma) The protein concentration was
deter-mined using the Bradford assay (Bio-Rad) Lysis were
running in a 8-15% sodium dodecyl
sulfate-polyacryla-mide electrophoresis (SDS-PAGE) gel, transferred to
PVDF membranes (Millipore), and incubated with
anti-bodys against CDKN2A, cyclin D1, total
retinoblas-toma protein (tRb), phosphorylated Rb protein (pRb),
and actin (Cell Signal Technology) and visualized by
enhanced chemiluminescence plus (GE)
CDKN2A construct
Full-length human CDKN2A cDNA was amplified by
PCR from a human fetal brain cDNA library
(Invitro-gen) by using primers contained restriction enzyme
cleavage sites (EcoRI and BamH I), and cloned into pcDNA3.1 vector (Invitrogen)
Small interfering RNA (siRNA) knockdown of CDKN2A Transient silencing of the CDKN2A gene was achieved using a pool of four siRNA duplexes (ONTARGETplus SMARTpool, Dharmacon) The target sequences were as follows: 5’-GATCATCAGTCACCGAAGG-3’, 5’-AAA-CACCGCTTCTGCCTTT-3’, 5’- TAACGTAGATA-TATGCCTT-3’, and 5’-CAGAACCAAAGCTCAAATA-3’
A mixture of four nontargeting siRNA duplexes was used
as a negative control (ON-TARGETplus Nontargetingv-Pool, Dharmacon) Transfections of H4 and HS-683 cells were performed using the Lipofectamine Plus transfection reagent (Invitrogen) according to the manufacturer’s instructions The efficiency of CDKN2A knockdown was detected by western blot 48 h after transfection
Colony formation assay and growth curves All glioma cells were transfected using Lipofectamin Plus (Invitrogen) in accordance with the procedure recommended by the manufacturer Forty-eight hours after tansfection, the cells were replated in 10 cm2 plates and maintained in selection medium containing 800μg/
ml of G418 (GIBCO) Cultures were replated in the den-sities of 1 × 103, 5 × 102, or 2.5 × 102on 10 cm2 plates
in triplicates and maintained for 2 weeks The neoresis-tant colonies were fixed with methanol, stained with Giemsa, and counted The number of colonies on the control dishes (transfected with pcDNA3.1 vector) was used as the 100% in this assay
The cells were transfected with pcDNA3.1 or CDKN2A using Lipofectamin Plus A mixed clones cells were obtained after G418 (800 μg/ml) selection for 1 week Growth curves were generated by plating 104cells
in the DMEM medium for 24, 48 72 and 96 hours in quadruples The cells were harvested with trypsin and counted at intervals
Statistical analyses Levels of CDKN2A are expressed as arithmetic means ± 95% confidence interval, statistical analysis was
Table 1 Summary of the pathological classification of glioma in index patients
Glioma classification WHO grade Male/Female N Age(years)
Pilocytic Astrocytoma(PA) I 3/1 4 27.1 ± 10.3
Astrocytoma(A) II 11/5 16 47.2 ± 6.9
Oligodendroglioma(O) II 3/3 6 54.8 ± 9.2
Low-Grade glioma 17/9 26 48.3 ± 9.1
Anaplastic Astrocytoma(AA) III 6/3 9 44.2 ± 10.7
Anaplastic Oligodendroglioma(AO) III 4/1 5 47.9 ± 5.4
Glioblastoma Multiforme(GBM) IV 16/5 21 55.3 ± 9.5
High-Grade glioma 26/9 35 52.2 ± 9.8
Trang 3performed using the Mann-Whitney U test All of
results are expressed as mean ± SD Values, statistical
analysis for the multiplicity was conducted using
ANOVA or Student’s t-test, where appropriate The
results were considered to be statistically significant
when P values were < 0.05
Results
Expression levels of CDKN2A in patients with malignant
gliomas and glioma cell lines
All of tumors were categorized based on the
histopatho-logic diagnosis Tumor samples were reevaluated by a
neuropathologist to confirm the diagnosis and were
graded using the World Health Organization criteria
Twenty-six tumors were classified as Low- Grade glioma (Grade I and II), and thirty-five tumors were graded High-Grade glioma (Grade III and IV) The stage of pri-mary tumors as well as further patient characteristics are shown in Table 1
CDKN2A is an important positive regulator of the cyclin-Rb signaling pathway involved in carcinogenesis
of glioma To confirm the role of CDKN2A in gliomas,
we detected the levels of CDKN2A expression in 61 glioma tissues by immunohistochemstry (IHC) (Figure 1A, C) and western blot (Figure 1B) Our results show that the expression levels of CDKN2A in high-grade glioma tissues were significant lower than that in low-grade glioma tissues Decreased CDKN2A in high-low-grade
Figure 1 The expression level of CDKN2A was associated with grade of gliomas Immunohistochemistry of CDKN2A in low-grade glioma (A), and grade glioma(B) Magnification, × 200 Immunohistochemistry statistical analysis results were shown low-grade gliomas v.s high-grade gliomas, p < 0.01 (B) Expression of CDKN2A was detected by western blot in low-high-grade glioma tissues and hig-high-grade glioma tissues 1-8: tissues from difference patients (C) Expression of CDKN2A protein in glioma cell lines (D) Note that H4 and HS-683 are low-grade glioma cell lines and the others were high-grade glioma cell lines Actin as loading control.
Trang 4glioma indicated that CDKN2A may be involved in
malignant glioma carcinogenesis We also detected the
expression of CDKN2A in high (T98G, U251-MG,
U87-MG, A172, SW1736, U118-MG and U138-MG) and low
grade glioma cells (H4 and HS-683) The result shows
that the high grade glioma cells have a lower levels of
CDKN2A than that of low-grade glioma cells, which in
consistent with glioma tissues from patients (Figure 1E)
Reconstitution CDKN2A suppresses colony-forming ability and growth rate of human malignant gliomas cells The molecular function of CDKN2A in tumor cells is a subject of considerable investigation, and it is still not clear To investigate whether anti-tumor effect of CDKN2A are affected by exogenous CDKN2A, various glioma cells were transfected with CDKN2A As shown
in Figure 2, CDKN2A potently inhibited colony-forming
A
0
20
40
60
80
100
120
T98G
＊
A
0 20 40 60 80 100 120
U87-MG
A
0 20 40 60 80 100 120
U251-MG
＊＊
＊
A
0
20
40
60
80
100
120
SW1783
A
0 20 40 60 80 100 120
A172
＊＊
＊
A
0 20 40 60 80 100 120
U118-MG
A
0 20 40 60 80 100 120
U138-MG
＊
＊
Figure 2 Effect of CDKN2A on colony-forming ability of human glioma cells CDKN2A suppresses colony-forming ability of human glioma cells All assays performed in triplicate The results were present by mean ± SD * P < 0.05, **P < 0.01 (Student ’s t-test) in all cases All
experiments were performed in triplicate.
Trang 5activity in various glioma cell lines Meanwhile,
Trans-fection of CDKN2A into glioma cells resulted in a
reduction in the rate of cell growth (Figure 3)
More-over, siRNA knockdown was performed in some
low-grade glioma cell lines (H4 and HS-683) When the
expression of CDKN2A interfered effectively, the cell
growth accelerates Our results indicated that
suppres-sing the expression of CDKN2A was able to promote
the low grade gliomas to high grade gliomas (Figure 4B
and 4C)
Antitumour effect of CDKN2A is Cyclin D1-dependent
To determine the role of the CDKN2A-Cyclin-Rb
path-way in glioma, Western blot analysis was used to detect
changes in expression of cell cycle regulatory proteins
Overexpression of CDKN2A had same effects on the
CDKN2A-Cyclin-Rb pathway proteins in various cell
lines (Figure 4) After overexpression of CDKN2A in
glioblastoma cell lines T98G, U87-MG and SW1783
MG, the expression of cyclin D1 was decreased The
phosphorylation of Rb protein (pRb) was also decreased
in all cell lines, but the level of total Rb was not
markedly reduced as phosphorylation of pRb In con-trast, we observed elevated levels of cyclin D1 and pRb when CDKN2A was knockdown However, flavopiridola,
an available cyclin D1 inhibitor [10,11] reserved the accelerated cell growth and the increased phosphoryla-tion of pBb induced by CDKN2A knockdown in low-grade glioma cells (Figure 4B, C and Figure 5B) More-over, a higher expression of Cylin D1 was observed in high-grade tumor tissues than that of low-grade tumor tissues (Figure 5C) The expression of Cylin D1 reversely correlates with CDKN2A expression in patients glioma tissues These results suggest that antitumour effect of CDKN2A is cyclin D1-dependent
Discussion
Genome-wide association study identifies that CDKN2A was a susceptibility loci for glioma [12] It was reported that CDKN2A be mutated and deleted in various human tumors, including more than 70% of human glioma cell lines and glioblastoma [13-16] In this study,
we identify that expression of CDKN2A was associated with grade of glioma in 61 patients with malignant glioma and glioma cells Lower level of CDKN2A was correlation with a worse prognosis Moreover, overex-pression of CDKN2A suppresses colony-forming ability and cell growth of various giloma cell lines It indicated that the level of CDKN2A expression may present the feedback mechanisms of the cell cycle in the malignant cell populations Subsequently, we investigated the effect
of CDKN2A on cell cycle by overexpression of CDKN2A in vitro Overexpression of CDKN2A sup-presses colony-forming ability and growth rate of human malignant glioma cells However, knockdown of CDKN2A promotes the low grade gliomas to high grade gliomas
There are three major pathways affected in a high per-centage of glioblastomas: receptor tyrosine kinase signal-ing, TP53 signaling and the pRB tumor suppressor pathway [6,17] The receptor tyrosine kinase (RTK) sig-naling pathway was involved in the translation of growth factor signals into increased proliferation and survival The altered genes in the RTK pathway include EGFR, PTEN, PIK3CA, RAS and TP53 signaling was important
in apoptosis, cellular senescence and cell cycle arrest in response to DNA damage Two TP53 inhibitors, MDM2 and MDM4, mediated the ubiquitinylation and degrada-tion of TP53 Also, the CDKN2A locus was frequently deleted or inactivated in glioblastomas and was involved
in both the TP53 pathway and pRB pathway The pRB
is a major protein involved in cell cycle progression from G1 to S phase CDK4, CDK6 and the hypopho-sphorylated state pRB bind to the transcription factor E2F, thereby preventing cell cycle progression Conver-sely, CDKN2A/CDKN2AINK4A, CDKN2B and
SW1738
0h 24 h 48 h 72 h 96 h
4000
6000
8000
10000
pcDNA3.1 CDKN2A
U87-MG
0h 24 h 48 h 72 h 96 h
4000
5000
6000
7000
8000
pcDNA3.1 CDKN2A
＊
＊＊
＊＊
＊＊
＊
A
B
Figure 3 Effect of CDKN2A on cell growth CDKN2A reduced the
growth of U87-MG (A) and SW1738 (B) glioma cell lines U87-MG
and SW1738 were transfected with pCDNA 3.1 vector and CDKN2A
respectively A mixed clones cells were obtained after G418 (800
μg/ml) selection for 1 week Growth curve experiment was
performed The results were present by mean ± SD * P < 0.05, **P
< 0.01 (Student ’s t-test) in all cases All experiments were performed
in triplicate.
Trang 6Figure 4 Konckdown of CDKN2A promotes the low grade gliomas to high grade gliomas Western blot analysis revealed a markedly decreased expression of CDKN2A after tranfecting a pool of four siRNA duplexes for CDKN2A in HS-683 and H4 cell lines(A) Knockdown of CDKN2A accelerates the growth of HS-683 (B) and H4 (C) glioma cell lines However, flavopiridola, a cyclin D1 inhibitor, can reverse the
accelerated cell growth both of HS-683 and H4 cell lines.
Figure 5 CDKN2A negatively regulated pRb and down-regulated level of cell cycle regulatory protein cyclin D1 Western blot analysis revealed a markedly lower phosphorylation of pRb and expression of cyclin D1 in T98G, U87-MG and SW1783 glioma cell lines transfected with CDKN2A (A) However, knockdown of CDKN2A increased the phosphorylation of pRb and cyclin D1 in H4 glioma cell line Moreover, a cyclin D1 inhibitor flavopiridol blocked the elevated phosphorylation of pRb and the expression of cyclin D1 induced by CDKN2A knockdown (B).
Increased cyclin D1 also detected in high-grade gliomas tissues comparing low-grade gliomas tissues (C) Three independent experiments were preformed A representative result was shown pRb, phosphorylated Rb; tRb, total Rb Actin as a loading control.
Trang 7CDKN2C, inhibit the different CDKs and are frequently
inactivated in GBM The CDKN2A acts as a
cyclin-dependent kinase inbibitor, inbibiting the binding of the
CDK4 protein to cylclin D1 and thus preventing
phos-phorylation of the Rb protein and arresting the cell
cycle in the G1phase [18,19] Cyclin D1 overexpression,
CDKN2A loss, and pRb inactivation play a key role in
glioma tumorigenesis [20-22] The results indicated that
overexpression CDKN2A has the potential to be
devel-oped into a future treatment for glioma patients
Conclusions
Our study suggests that CDKN2A as a malignant
glio-mas suppressor gene, appears to be useful for predicting
behaviour of high-grade malignant gliomas
CDKN2A-Cyclin-Rb pathway plays a key role on malignant
glio-mas formation and that therapeutic targeting of this
pathway may be useful in malignant gliomas treatment
Abbreviations
CDKN2A: cyclin-dependent kinase inhibitor 2A; Rb: retinoblastoma protein;
pRb: phosphorylation of Rb protein; tRb: total Rb protein; IHC:
immunohistochemstry; RTK: receptor tyrosine kinase.
Authors ’ contributions
WL and YL carried out most of the experiments listed in this study; WL
drafted the manuscript; BW and LG designed the project and drafted the
manuscript All authors read and approved the final manuscript
Competing interests
The authors declare that they have no competing interests.
Received: 10 April 2011 Accepted: 15 August 2011
Published: 15 August 2011
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doi:10.1186/1756-9966-30-76 Cite this article as: Liu et al.: Downregulation of CDKN2A and suppression of cyclin D1 gene expressions in malignant gliomas Journal
of Experimental & Clinical Cancer Research 2011 30:76.
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